Type 2 diabetes mellitus (T2DM) and (Ob) are major public health concerns that are increasing at Type obesity alarming rates. Insulin resistance (IR) in skeletal muscle is central to the pathophysiology of T2DM and obesity. A phenotype of muscle IR that is well described is reduced glucose utilization during insulin-stimulation, a reduction associated with impaired glucose transport, reduced glycogen synthesis and impaired insulin signal transduction; for brevity, we will refer to this phenotype as IR-Glu. This project, (DK049200), has been productive in examining an aspect of muscle IR that is additional to IR-Glu, namely an impaired reliance upon fat oxidation (FATOX) during fasting, and has been productive in elucidating cellular mechanisms responsible for impaired FATOX. It has been especially intriguing to examine the relationship between impaired FATOX during fasting conditions and IR-GIu. Physiologically, impaired FATOX during fasting is correlated with severity of IR-Glu, and this association is further typified by inflexibility in rates of FATOX in the transition between fasting and insulin-stimulated conditions, a sharp contrast to what occurs in insulin sensitive skeletal muscle. We believe that our work has begun to elucidate a shared cellular mechanism for IR-Glu and impaired FATOX. One current concept is that intra-myocellular lipid content (IMLC) might be a mechanistic link between these two facets of muscle IR; wherein decrease FATOX yield increase IMLC yield IR-Glu. Whether it is IMLC (largely tdglyceride) that induces IR-Glu or whether this is a marker and other lipid moieties such as long chain acylCoA, DAG, ceramide, or altered fatty acid composition in phospholipids mediate IR is an active and important area of these investigations. Our principle effort, while now wholly removed from the above postulate, has been, however, to focus more upon potential cellular mechanisms that lead to decrease FATOX and increase IMLC. Through a series of studies in the first two funding periods, we have found the muscle in T2DM and Ob has a reduced capacity for oxidative phosphorylation (OXPHOS). Recent human gene array studies strongly support this concept. Our overall goal in this application for renewal of funding will be to examine the hypothesis that decrease OXPHOS yield IR-Glu. One possibility is that decrease OXPHOS yield decrease FATOX increase IMLC yield IR-Glu. Our studies will examine this hypothesis, but too, we propose based upon exciting preliminary data that there is a plausible more direct link. We have made a recent and novel observation that in T2DM and in Ob, to a lesser extent, there is a particularly severe reduction of OXPHOS in sub-sarcolemmal population of mitochondria (SubSarco-M), the sub-population near the cell surface that provide ATP for ion pumps, signal transduction, and substrate uptake. We propose integrated physiology studies in humans and in the context of weight loss and physical activity interventions, to assess in vivo substrate metabolism by muscle and to assess muscle OXPI-IOS, IMLC, and insulin signaling, as well as sites of damage in mitochondria and responsiveness of mitchondrial biogeneisis in IR.